Specific Aims: Lack of motivation to exercise is the hallmark of obesity and type2 diabetes (T2D). High fat diet (HFD) is the major cause of obesity and T2D that impair contractility of cardiomyocytes leading to heart failure. The stimulation of 2-adrenergic receptors (2-AR) induces contractility and mitigates cardiac dysfunction. Elevated level of homocysteine (Hcy) called hyperhomocysteinemia (HHcy, a co-morbid condition) is associated with heart failure in diabetes. Hcy competes with and antagonizes 2-AR. The long term goal of the project is to understand the role of 2- AR and Hcy axis in diabetes. HHcy is ameliorated by conversion of Hcy to H2S (an anti-hypertensive, vasorelaxing, and anti-oxidant gas) by cystathionine synthase (CBS) and cystathionine ? lyase (CSE). In diabetes, the levels of CBS and CSE are suppressed that induces HHcy and thereby impairs formation of H2S. H2S is an inducer of AKT (an anti-oxidant involved in glucose metabolism) and AKT is down regulated in diabetes. We have shown that H2S induces a stimulatory G-protein couple receptor called Gs (inducer of 2-AR) in diabetic cardiomyocytes. On the other hand, HHcy attenuates Gs. However, exercise mitigates HHcy and up regulates 2-AR in T2D. Also, exercise and salbutamol (2-AR agonist) have synergistic effect on mitigation of contractile dysfunction in diabetic cardiomyocytes. However, the underlying mechanism is unclear. We have shown that in diabetes, HHcy activates MMP9 that induces cardiac fibrosis and impairs myocardial contractility. Also, microRNA-133a (miR-133a) that regulates cardiac fibrosis and hypertrophy is attenuated in diabetic hearts. Our preliminary studies show that HHcy (CBS+/- mice) and HFD treatment attenuate myocardial 2-AR and miR-133a, whereas exercise and H2S mitigate this attenuation. Exercise and salbutamol have synergistic effect on induction of AKT and Gs in diabetes. The transgenic expression of 2-AR (2-ARTg) mitigates HFD induced cardiac fibrosis. Also, ablation of MMP9 gene ameliorates HFD induced contractile dysfunction. Both HFD treated WT and CBS+/- mice have left ventricle dysfunction and impaired contractility, which is mitigated by exercise training. HHcy and HFD also down regulates sarco-endoplasmic reticulum ATPase 2a (serca2a; regulates calcium flux during muscle contraction), which is improved by exercise and H2S. The central hypothesis of the proposal is that in HFD induced diabetes, the intolerance to exercise is, in part, due to attenuation of 2-AR and Gs by HHcy that down regulates AKT and miR-133a and induces MMP9 causing myocytes contractile dysfunction. The exercise and H2S induce 2-AR and Gs by mitigating HHcy and ameliorate contractile dysfunction in diabetes (Figure 1). We will test this hypothesis by following three specific aims: Specific Aim#1: To determine whether the 2-AR is attenuated by hyperhomocysteinemia in diabetes and exercise and H2S mitigate this attenuation. Hypothesis: In diabetes, 2-AR is down regulated and Hcy is elevated, and exercise and H2S induce 2-AR and decrease Hcy level. Specific Aim#2: To determine whether the AKT and miR-133a are attenuated and MMP9 is induced by hyperhomocysteinemia in diabetes and exercise and H2S ameliorate this attenuation and induction. Hypothesis: In diabetes, the elevated level of Hcy inhibits AKT and miR-133a and induces MMP9. Exercise and H2S induce AKT and attenuate MMP9 by decreasing Hcy level in diabetic hearts. Specific Aim#3: To determine whether the contractility of cardiomyocytes is impaired by hyperhomocysteinemia in diabetes and exercise and H2S mitigate the contractile dysfunction. Hypothesis: In diabetes, Hcy and MMP9 are elevated while 2-AR and AKT are attenuated leading to contractile dysfunction, and exercise and H2S ameliorate these levels and mitigate contractile dysfunction. These studies will elucidate the mechanism of homocysteine-mediated attenuation of 2-AR in diabetes and cardio-protective role of 2-AR agonist, exercise and H2S in diabetic complications.